Peptide that regulates fat metabolism and method for regulating fat metabolism

ABSTRACT

The present disclosure relates to a peptide for regulating fat metabolism and a method of regulating fat metabolism in an individual. The peptide is a variant of a SEQ ID NO: 2 having an amino acid sequence of YLYQWLGAPVPYPDPLEP, the variant comprises peptides selected from the group consisting of: (a) a first amino acid sequence consisting of the SEQ ID NO: 2, no more than 5 amino acids inserted to a C-terminal of the SEQ ID NO: 2, and no more than 5 amino acids inserted to a N-terminal of the SEQ ID NO: 2; (b) a second amino acid sequence consisting of a modified SEQ ID NO: 2; and (c) a third amino acid sequence consisting of at least 6 contiguous amino acids of the SEQ ID NO: 2 or the modified SEQ ID NO: 2.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is part of theapplication and is provided in text in the form of an ASCII text file inlieu of a paper copy, and is hereby incorporated by reference into thespecification. The name of the text file containing the Sequence listingis PeptideSequenceListing. The size of the text file is 4 kilobytes, wascreated on Sep. 8, 2021, and is being electronically submitted viaEFS-Web.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.16/572,584 filed on Sep. 16, 2019, which is a continuation-applicationof PCT Application No. PCT/CN2017/076886, filed on Mar. 16, 2017.

TECHNICAL FIELD

The present disclosure relates to the field of biomedicine, and inparticular to a peptide for regulating fat metabolism.

BACKGROUND

Obesity is a major and growing health problem worldwide. Obesity is alsoa risk factor for the development of many common diseases such asatherosclerosis, hypertension, type 2 diabetes, dyslipidemia, coronaryheart disease, osteoarthritis and various malignancies. It also causesmore serious problems by reducing exercise capacity and quality of life.Occurrence of the obesity and the diseases caused by the obesity aregrowing in all developed countries.

Fatty liver refers to a lesion caused by excessive accumulation of fatin hepatocytes. There are many causes of the fatty liver, such asalcoholism, unreasonable diet, and sedentariness. It witnessesincreasing incidence in China and constitutes a threat to people'shealth.

However, the current drugs and methods for treating the obesity and thediseases caused by the obesity, including nonalcoholic fatty liver(NAFLD), have certain deficiencies, so it needs to develop medicines andmethods with better efficacy and lower side effects for the treatment ofthe obesity and the NAFLD.

Osteocalcin (OCN) is a vitamin K-dependent calcium-binding protein and anon-collagen acidic glycoprotein synthesized and secreted byosteoblasts, the vitamin K-dependent glutamate residue in its moleculeis an important functional group for the OCN to bind with Ca²⁺.

SUMMARY OF THE DISCLOSURE

In order to provide a more effective treatment for the obesity-inducedNAFLD and other obesity-induced diseases, the present disclosure mayprovide a peptide for regulating fat metabolism, wherein the peptide isa variant of a SEQ ID NO: 2 having an amino acid sequence ofYLYQWLGAPVPYPDPLEP, the variant comprises peptides selected from thegroup consisting of: (a) a first amino acid sequence consisting of theSEQ ID NO: 2, no more than 5 amino acids inserted to a C-terminal of theSEQ ID NO: 2, and no more than 5 amino acids inserted to a N-terminal ofthe SEQ ID NO: 2; (b) a second amino acid sequence consisting of amodified SEQ ID NO: 2, no more than 5 amino acid sequence inserted to aC-terminal of the modified SEQ ID NO: 2, and no more than 5 amino acidsequence inserted to a N-terminal of the modified SEQ ID NO: 2, whereinthe modified SEQ ID NO: 2 is obtained by: deleting the Y (Tyr) at aposition 3 of the SEQ ID NO: 2 or substituting the position 3 of the SEQID NO: 2 to be Asn or Asp; deleting the Q (Gln) at a position 4 of theSEQ ID NO: 2 or substituting the position 4 of the SEQ ID NO: 2 to beAsn, His, Pro, or Ser; deleting the W (Trp) at a position 5 of the SEQID NO: 2 or substituting the position 5 of the SEQ ID NO: 2 to be Gly;deleting the L (Leu) at a position 6 of the SEQ ID NO: 2; substitutingthe P (Pro) at a position 9 of the SEQ ID NO: 2 to be Ser; substitutingthe V (Val) at a position 10 of the SEQ ID NO: 2 to be Ala; substitutingthe Y (Tyr) at a position 12 of the SEQ ID NO: 2 to be Ser; substitutingthe P (Pro) at a position 15 of the SEQ ID NO: 2 to be Thr; and a totalnumber of positions being substituted and deleted in the second aminoacid sequence defined in (b) is less than 4; and (c) a third amino acidsequence consisting of at least 6 contiguous amino acids of the SEQ IDNO: 2 or the modified SEQ ID NO: 2.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows body weight comparison of a model of C57BL/6 mice having adiet-induced-obesity and nonalcoholic fatty liver disease (DIO-NAFLD)generated by feeding with high fat diet (HFD) for 12 weeks with mice ofa normal control group fed with normal diet (ND).

FIG. 2A shows an effect of daily intraperitoneal (i.p.) injection ofvarious concentrations of insulin secretion association peptide 1 (SEQID NO.17) and OCN (mouse OCN) for 6 weeks on a size of an epididymal fatpad of a DIO-NAFLD mouse fed with high-fat diet, comparing with that ofa mouse with HFD only (a HFD control group) and a mouse with ND only (aND control group).

FIG. 2B shows average mass of the epididymal fat pads of the mice in SEQID NO.17-treated groups, in an OCN-treated group, in the HFD controlgroup, and in the ND control group (according to statistical analysis,*: P<0.05, compared with the HFD control group).

FIG. 2C shows hematoxylin and eosin (H&E) staining of an epididymal fatpad tissue slice obtained from each epididymal fat pad shown in FIG. 2A.

FIG. 2D shows average surface areas of each single adipocyte calculatedbased on the H&E staining of the epididymal fat pad tissue slice underdouble-blind conditions (according to statistical analysis, ***:P<0.001, compared with the HFD control group).

FIG. 3A shows representative images of liver appearances obtained fromfive mice selected from two groups with daily i.p. injection of variousconcentrations of SEQ ID NO.17, from a group with daily i.p. injectionof OCN, from the HFD control group, and from the ND control group.

FIG. 3B shows H&E staining of liver tissue slices obtained from thelivers shown in FIG. 3A.

FIG. 3C shows surface areas of accumulated Oil Red 0 dye from livercells of the mice from each group after Oil Red 0 staining underdouble-blind conditions, wherein each group includes six mice (accordingto statistical analysis, *: P<0.05, **: P<0.01, and ***: P<0.001,compared with the HFD control group).

FIG. 4A shows an effect of daily i.p. injection of variousconcentrations of SEQ ID NO.17 and OCN (mouse OCN) for 6 weeks on alevel of alanine aminotransferases (ALT) in blood of the DIO-NAFLD micefed with HFD, comparing with that in the mice of the HFD control group,wherein each group includes six mice (according to statistical analysis,*: P<0.05 and **: P<0.01, compared with the HFD control group).

FIG. 4B shows an effect of daily i.p. injection of variousconcentrations of SEQ ID NO.17 and OCN (mouse OCN) for 6 weeks on alevel of alkaline phosphatases (ALP) in blood of the DIO-NAFLD mice fedwith HFD, comparing with that in the mice of the HFD control group,wherein each group includes six mice (according to statistical analysis,*: P<0.05, compared with the HFD control group).

FIG. 4C shows an effect of daily intraperitoneal injection of variousconcentrations of SEQ ID NO.17 and OCN (mouse OCN) for 6 weeks on alevel of aspartate aminotransferases (AST) in blood of the DIO-NAFLDmice fed with high-fat diet, comparing with that in the mice of the HFDcontrol group and that in the mice of the ND control group, wherein eachgroup includes six mice (according to statistical analysis, *: P<0.05,compared with the HFD control group; and #: P<0.05 and ##: P<0.01,compared with the ND control group).

FIG. 4D shows an effect of daily i.p. injection of variousconcentrations of SEQ ID NO.17 and OCN (mouse OCN) for 6 weeks on alevel of serum total cholesterols (TC) of the DIO-NAFLD mice fed withHFD, comparing with that in the mice of the HFD control group, whereineach group includes six mice (according to statistical analysis, *:P<0.05 and **: P<0.01, compared with the HFD control group).

FIG. 4E shows an effect of daily i.p. injection of variousconcentrations of SEQ ID NO.17 and OCN (mouse OCN) for 6 weeks on alevel of low density lipoprotein (LDL) of the DIO-NAFLD mice fed withHFD, comparing with that in the mice of the HFD control group and thatin the mice of the ND control group, wherein each group includes sixmice (according to statistical analysis, *: P<0.05, **: P<0.01, and ***:P<0.001, compared with the HFD control group; and ###: P<0.001, comparedwith the ND control group).

FIG. 4F shows an effect of daily i.p. injection of variousconcentrations of SEQ ID NO.17 and OCN (mouse OCN) for 6 weeks on alevel of high density lipoprotein (HDL) of the DIO-NAFLD mice fed withHFD, comparing with that in the mice of the HFD control group and thatin the mice of the ND control group, wherein each group includes sixmice (according to statistical analysis, *: P<0.05 and ***: P<0.001,compared with the HFD control group; and ###: P<0.001, compared with theND control group).

FIG. 5A shows frozen sections of jejunum segments near duodenumsegments, obtained from ND-fed wild-type C57BL/6 mice gavaged withsterilized olive oil followed by i.p. injection of SEQ ID NO.17 and OCNbefore sacrificing and from ND-fed wild-type C57BL/6 mice in controlgroups, stained with oil red O and counterstained with hematoxylin.

FIG. 5B shows quantification of oil red O positive areas shown in FIG.5A by an ImageJ software under double-blind conditions, and shows aratio of the positive areas to a total area of intestinal villi(according to statistical analysis, ***: P<0.001, N=3, compared with theratio for the mice treated with normal saline and the sterile oliveoil.).

FIG. 6A shows overexpression of human GPRC6A (hPGRC6A) in Hela cells.

FIG. 6B shows binding of SEQ ID NO.17, OCN and OCN-22 to cell membranesof the Hela cells with hGPRC6A overexpressed and binding of SEQ ID NO.2,human OCN (hOCN) and human OCN-22 (hOCN-22) to the cell membranes of theHela cells with hGPRC6A overexpressed.

FIG. 6C shows effects of Cy-5-labeled OCN (Cy5-OCN), Cy-5-labeled OCN-22(Cy5-OCN22), and Cy-5-labeled SEQ ID NO.2 (Cy5-SEQ ID NO.2) on GPRC6Ainternalization in the Hela cells with hGPRC6A overexpressed.

FIG. 7A shows effects of gavaged OCN, SEQ ID NO.17, SEQ ID NO.2, and SEQID NO.3 to mice fed with HFD and ND for 7 weeks on body weights of themice.

FIG. 7B shows levels of triglyceride in feces of the mice in an SEQ IDNO.17-treated HFD group, in an SEQ ID NO.2-treated HFD group, in an SEQID NO.3-treated HFD group, in an OCN-treated HFD group, in the HFDcontrol group, and in the ND control group.

FIG. 8 shows comparison of the sequences of SEQ ID NO.4, SEQ ID NO.1,SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.6, SEQ ID NO.8, SEQ IDNO.9, SEQ ID NO.10 and SEQ ID NO.11 from various species.

FIG. 9 shows effects of gavaged SEQ ID NO.15, SEQ ID NO.16, and SEQ IDNO.14 on a ratio of areas of the jejunum containing fat to a total areaof intestinal villi of ND-fed wild-type C57BL/6 mice (according tostatistical analysis,***: p<0.001, and n=3, compared with the micetreated with “normal saline+the olive oil”).

DETAILED DESCRIPTION

As used herein, the term “peptide that regulates fat metabolism” (alsoreferred to as “insulin secretion association peptide (ISAP)” refers toan OCN originated or derived peptide that may regulates energymetabolism and variants of the OCN originated or derived peptide.

The term “conservative amino acid substitution” as used herein refers tothe substitution of the original amino acid sequence with another aminoacid residue having similar properties. For example, lysine residues,arginine residues, and histidine residues may be similar in having basicside chains. Furthermore, aspartic acid residues and glutamic acidresidues may be similar in having acidic side chains. Furthermore,asparagine residues, glutamine residues, serine residues, threonineresidues, tyrosine residues and cysteine residues may be similar inhaving uncharged polar side chains, and glycine residues, alanineresidues, valine residues, leucine residues, isoleucine residues,proline residues, tryptophan residues, phenylalanine residues, andthionine residues may be similar in having non-polar side chains.Further, tyrosine residues, phenylalanine residues, tryptophan residues,and histidine residues may be similar in having aromatic side chains.Thus, it will be apparent to the skilled in the related art that aminoacid substitutions made in groups of amino acids having similarproperties as described above do not cause any change in properties.

Full names and abbreviations of the amino acids used in the presentdisclosure are listed hereafter:

Three-letter One-letter Full name abbreviation abbreviation Serine Ser SThreonine Thr T Asparagine Asn N Glutamine Gln Q Tyrosine Tyr Y CysteineCys C Aspartic acid Asp D Glutamate Glu E Histidine His H Lysine Lys KArginine Arg R Glycine Gly G Alanine Ala A Valine Val V Leucine Leu LIsoleucine Ile I Phenylalanine Phe F Methionine Met M Proline Pro PTryptophan Trp W

The term “disease associated with abnormal fat metabolism” as usedherein refers to a disease characterized by fat metabolism disorder or acomplication thereof caused by genetics or environment or both, such asbut not limited to obesity, Type 2 diabetes, NAFLD, insulin resistance,hypertriglyceridemia, hypercholesterolemia, atherosclerosis, andcoronary heart disease.

The term “non-alcoholic fatty liver disease” as used herein refers to aclinicopathologic syndrome characterized by excessive deposition of fatin hepatocytes due to factors other than alcohols.

The term “insulin resistance” as used herein refers to a state in whichcells cannot effectively “burn” glucose as insulin-mediated glucoseuptaken and consumption is reduced. When insulin resistance is at a highlevel, the body may produce excessive insulin, leading to high bloodpressure, abnormal lipidemia, heart diseases and diabetes. Especially ina case of type 2 diabetes, insulin may not function normally becausemuscles and adipose tissues cannot recognize an increase of the insulin.

Terms not explicitly defined herein have meanings as commonly understoodby the skilled in the related art.

EMBODIMENTS

The Dulbecco's Modified Eagle Medium (DMEM) used in the presentdisclosure is purchased from Sigma. 10% of Fetal Bovine Serum (FBS), 1%of non-essential amino acids, 1 g of glucose, 0.75 g of sodiumbicarbonate, 0.1 g of bovine serum albumin and 1.5 ml of4-hydroxyethylpiperazineethanesulfonic acid (HEPES) may be supplementedinto 500 ml of the media for cell culturing.

3T3L1 cells may be purchased from American Type Culture Collection(ATCC).

ISAP₁, ISAP₂, ISAP₃, ISAP₄, ISAP₅, and ISAP₆ may be manmade synthesized.Sequences of these peptides are listed in a following table.

TABLE I Amino acid sequences of various ISAPs synthesized Peptide SEQ IDAmino Acid Sequence ISAP₁ SEQ ID Tyr-Leu-Gly-Ala-Ser-Val-Pro-Ser- NO: 17Pro-Asp-Pro-Leu-Glu-Pro-Thr ISAP₂ SEQ IDTyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala- NO: 2 Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro ISAP₃ SEQ ID Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala- NO: 3Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu- Glu-Pro-Arg ISAP₄ SEQ IDSer-Val-Pro-Ser-Pro-Asp-Pro-Leu- NO: 15 Glu-Pro ISAP₅ SEQ IDPro-Ser-Pro-Asp-Pro-Leu-Glu-Pro NO: 16 ISAP₆ SEQ IDPro-Asp-Pro-Leu-Glu-Pro NO: 14

All the animal experiments have been approved by the Animal EthicsCommittee of the Shenzhen Institutes of Advanced Technology (SIAT) ofthe Chinese Academy of Sciences (CAS), in accordance with therequirements of the Ethics Committee.

Embodiment 1

Study of ISAP₁ Functions

S110: a diet-induced obesity and nonalcoholic fatty liver disease(DIO-NAFLD) mouse model by HFD feeding may be established.

Thirty specific pathogen free (SPF) level healthy 6-week-old maleC57BL/6 mice may be purchased from the Experimental Animal Center ofGuangdong Province. A body mass of each mouse may be 18 g to 22 g. Themice may be divided into two groups, housed in a SPF-grade animalfacility of SIAT. Six mice may be fed with ND (the ND contains 5% offat; 53% of carbohydrate; 23% of protein, having total calories of 25J/g) for 12 weeks to be a ND control group, and may be allowed to havefood and water ad lib. The other mice may be fed with HFD (the HFD maybe D12451, Research Diets, Inc.) for 12 weeks, and may be allowed tohave food and water ad lib. Physiological indexes of the mice may bedetected. A body weight of a mouse fed with the HFD may be 15% greaterthan that of a mouse fed with ND, suggesting the DIO-NAFLD mouse modelmay have been established successfully. In the present embodiment, anaverage body weight of the DIO-NAFLD mice may be more than 40 g.Comparison of average body weight of the mice of the established modelwith that of the mice in the ND control group is shown in FIG. 1. Thearrow indicates a time point at which the HFD be started.

S120: effect of i.p. injection of ISAP₁ on DIO-NAFLD mice may bestudied.

S121: effect of i.p. injection of ISAP₁ on epididymal fat pad may bestudied.

When the body weight of the each mouse fed with the HFD is more than 40g, and when the blood glucose level of the each mouse is above 10 mMol,each DIO-NAFLD mouse may be treated with i.p. injection of ISAP₁ for 6consecutive weeks. The DIO-NAFLD mice may be divided into 4 groups, 6mice in each group. ISAP₁ may be dissolved in normal saline solutioncontaining 0.01% of BSA. A dosage of 20 pmol/g of ISAP₁ may be i.p.injected into each DIO-NAFLD mouse in group #1 once a day, and a dosageof 2 pmol/g of ISAP₁ may be i.p. injected into each DIO-NAFLD mouse ingroup #2 once a day. OCN (mouse OCN protein) may be prepared andadministered similarly as the ISAP₁ at a dosage of 6 pmol/g to eachmouse in group #3. The mice in group #4 may be treated with normalsaline only as a HFD control group. After 6 weeks, the mice may beeuthanized by application of 95% CO2, and epididymal fat pads of 4 miceselected from each group and an epididymal fat pad of a mouse from theND control group may collected and weighed. Paraffin slices of eachepididymal fat pad may be prepared, and an area of the adipocytes may beobserved by microscopy.

Experimental results are shown in FIG. 2. FIG. 2A shows overallappearances of the epididymal fat pads of the mice in the ND controlgroup, in the HFD control group, and in the group receiving daily i.p.injection of ISAP₁ or OCN, wherein the administration of ISAP₁ maysignificantly reduce the size of the epididymal fat pads as comparedwith the HFD control group. FIG. 2B shows an average mass of theepididymal fat pads of the mice in each group, wherein theadministration of ISAP1 may significantly reduce the weight of theepididymal fat pad as compared with the HFD control group. FIG. 2C showsH&E staining of the epididymal fat pads shown in FIG. 2A, revealing thatthe administration of ISAP₁ may significantly reduce sizes of adipocytesas compared with the HFD control group. FIG. 2D shows average surfaceareas of each single adipocytes calculated from H&E stained slices underdouble-blind conditions, indicating that ISAP₁ may significantly reducethe sizes of the adipocytes. Statistical analysis may be performed tocompare with the HFD control group, wherein *: P<0.05, **: P<0.01, and***: P<0.001.

S122: effect of i.p. injection of ISAP₁ on the liver may be studied.

In S121, livers may be collected while collecting the adipose tissues,and appearance of the livers may be photographed. A part of livertissues may be taken for frozen sectioning, stained with oil red O formicroscopic observation, and then a same area may be quantitativelyanalyzed. Results are shown in FIG. 3. FIG. 3A shows representativeimages of liver appearances for five mice selected from each group. Thelighter the color is, the higher the fat content is contained. Treatmentof HFD and a carrier (HFD+carrier) may result in significant liver fataccumulation, and i.p. injection of ISAP₁ may significantly reduce theliver fat accumulation. FIG. 3B shows H&E staining of a liver sliceobtained from the livers shown in FIG. 3A, wherein a light colorrepresents intracellular stained fat. FIG. 3C shows surface areas ofaccumulated non-alcoholic fatty liver cells obtained after liver oil redO staining under double-blind conditions from the mice in each group. Itis shown that a proportion of fat in the liver of the mice in theISAP₁-treated group is decreased significantly as compared with that ofthe mice in the HFD control group, indicating that ISAP₁ may reduce thefat content in hepatocytes.

S123: the effect of i.p. injection of ISAP₁ on liver functions and bloodlipids in mice may be studied.

In S121, before sacrificing the mice, blood of each mouse may becollected from a tail vein and detected for a level of alanineaminotransferases (ALT), a level of alkaline phosphatases (ALP), a levelof aspartate aminotransferases (AST), a serum level of cholesterols, alevel of low-density lipoprotein (LDL), and a level of high-densitylipoprotein (HDL) using a Roche blood glucose meter (model cobas8000) asper manufacturer's instructions. Experimental results are shown in FIG.4. ISAP₁ may be effective in reducing the ALT, the ALP, the AST, thecholesterols, and the LDL even at the dosage of 2 pmol/g, comparing withthe HFD control group.

It can be seen from the above results that i.p. injection of ISAP₁ cansignificantly affect the fat metabolism in mice, reduce fat accumulationin hepatocytes and adipocytes, improve the fat metabolism, andeffectively alleviate progression of the NAFLD.

S130: binding of ISAP₁ to human GPRC6A (hGPRC6A) may be studied.

S131: overexpression of hGPRC6A in Hela cells may be established.

1) Cell plating: A Hela cell suspension may be plated at a density of1.6×105 cells/mL into a 6-well tray with 2 mL of DMEM per well,incubating at 37° C. and 5% CO2 for 24 hours.

2) A vector carrying hGPRC6A (pReceiver-M61) may be transfected into theHela cells for overexpression, using Lipofectamine 2000 (Invitrogen) asper manufacturer's instructions, and media containing the vector may bereplaced by normal media 4 hours later.

3) After the transfection, the cells may be cultured in the normal mediafor another 48 hours, and the normal media may be discarded. The cellsattached on a bottom of each well may be washed twice with sterile PBS,and 300 μl of TRIzol solution may be added into each well. Cellular RNAsmay be extracted as per RNAiso Plus (TaKaRa) instructions, and after aDNase treatment, may be reversely transcribed into cDNAs using DNAEngineand a SuperScript™ (Invitrogen, Canada) kit. The cDNAs may be used as atemplate, and real-time monitoring and analysis may be performed tofluorescent PCR products at various time points using a SYBR Green(Light Cycler Roche, Germany) method to detect expression levels of thehGPRC6A. If necessary, screening using puromycin may be performed toobtain a cell strain with stable expression of the hGPRC6A. The selectedcell strain may then be detected for its gene expression by qPCR. Asshown in FIG. 6A, hGPRC6A may be stably expressed in a large amount inthe selected Hela cells.

S132: experiments of binding various peptides of OCN to cell membranesof Hela cells with hGPRC6A overexpressed may be performed.

To detect the binding between the peptides and the hGPRC6A, afluorescent detection may be performed. A higher fluorescent intensitymay indicate a higher level of binding of the peptides to the hGPRC6A.Further, a competitive agonist may be added into the media tocompetitively inhibit the peptides from binding to the hGPRC6A, and thefluorescent intensity may be reduced. Experimental results are shown inFIG. 6B. Compared with OCN, affinity of the ISAP₁ binding to the hGPRC6Amay be consistent with that of the OCN binding to the hGPRC6A. AlthoughhOCN-22 may bind to hGPRC6A, but affinity between hOCN-22 and hGPRC6Amay be 10 times lower than that between ISAP₁ and hGPRC6A, suggestingthat ISAP₁ may be a core domain of OCN to interact with the receptorhGPRC6A.

S140: effect of acute gavage of ISAP₁ on intestinal fat absorption innormal mice may be studied.

Six-week-old male wild-type C57BL/6 mice may be divided into fourgroups, three in each group, three out of the four groups of mice may beintestinally perfused with 200 μl sterilized olive oil solution (Sigma),and the other one group of mice may be intestinally perfused with normalsaline solution to be a negative control group. After 30 minutes, twogroups of the mice treated with sterilized olive oil solution may bei.p. injected with OCN (6 pmol/g) and ISAP₁ (6 pmol/g) respectively, andthe other one group of the mice treated with sterilized olive oilsolution may be i.p. injected with normal saline solution to be a salinecontrol group. Further the mice of the negative control group solutionmay also be i.p. injected with normal saline solution to be the negativegroup. The mice of all four groups may be sacrificed 30 minutes later.Small intestine samples may be collected. The small intestine sample maybe taken from a duodenum to a caecum, divided into 3 segments of equallength. After washing with pre-cooled normal saline solution, a segmentclose to the duodenum may be frozen and sliced, stained with oil red Oand observed for fat absorption.

Results of the experiment are shown in FIG. 5. FIG. 5A shows jejunumsamples close to the duodenum segments, collected from four mice of eachgroup, being frozen and sliced, stained with oil red O, andcounterstained with hematoxylin. FIG. 5B shows oil red O-positive areasshown in FIG. 5A being quantified by the ImageJ software underdouble-blind conditions, and a ratio of the oil red O-positive area to atotal area of the intestinal villi in each sample may be calculated. Itmay be shown that, compared with the saline control group, the treatmentof ISAP₁ may effectively reduce the mouse intestinal absorption of theolive oil, and the ISAP₁-induced reduction in the olive oil absorptionmay be even more effective than the OCN treatment.

Six-week-old male wild-type C57BL/6 mice may be divided into 6 groups, 6mice in each group. Group #1 may be provided with ND, and groups #2 to#6 may be provided with HFD. Group #2 may be treated as a HFD controlgroup. Groups #3 to #6 may be experimental groups, and mice in groups #3to #6 may be gavaged with OCN, ISAP₁, ISAP₂, and ISAP₃ respectively at 2pmol/g of body weight for 7 weeks. Body weight of each mouse may bemonitored and measured once a week. Experimental results are shown inFIG. 7A. It may be shown from the figure that a significant weight lossoccurred for the mice in the ISAP₁-treated group as compared with themice in the HFD control group. *: P<0.05, **: P<0.01, ***: P<0.001.

Feces of each mouse may be collected in week 7, baked at 60° C. for 3days to ensure complete dryness. Then, 1 mg of the feces may becollected and soaked in 1 ml of a mixed solution consisting ofchloroform and methanol in a ratio of 2:1. The soaked feces may then bedisrupted with a tissue homogenizer and centrifuged to obtain asupernatant. The supernatant may be detected for triglyceride by theRoche blood biochemical analyzer. Experimental results are shown in FIG.7B. **: P<0.01, ***: P<0.001. Compared with the HFD control group, thecontent of triglyceride in the feces collected from the mice in theISAP₁-treated group may be significantly increased, indicating thatgavage of ISAP₁ may significantly reduce absorption of triglyceridesthrough intestinal tracts.

Embodiment 2

Study of ISAP₂ Functions

S210: binding of ISAP₂ to human GPRC6A may be studied.

S211: overexpression of hGPRC6A in Hela cells may be established.

1) Cell plating: A Hela cell suspension may be plated at a density of1.6×105/mL into a 6-well tray with 2 ml of DMEM media per well,incubating at 37° C. and 5% CO2 for 24 hours.

2) A vector carrying hPGRC6A (pReceiver-M61) may be transfected into theHela cells for overexpression, using Lipofectamine 2000 (Invitrogen) asper manufacturer's instructions, and the media containing the vector maybe replaced by normal media 4 hours later.

3) After the transfection, the cells may be cultured in the normal mediafor another 48 hours, and the media may be discarded. The Hela cellsattached to a bottom of each well of the tray may be washed twice withsterile PBS, and 300 μl of TRIzol solution may be added into each well.Cellular RNAs may be extracted as per RNAiso Plus (TaKaRa) instructions.After a treatment with DNase, the extracted cellular RNAs reverselytranscribed into cDNAs using DNA Engine and a SuperScript™ (Invitrogen,Canada) kit. The cDNAs may be used as a template, and real-timemonitoring and analysis may be performed to fluorescent PCR products atvarious time points using a SYBR Green (Light Cycler Roche, Germany)method to detect expression levels of hGPRC6A. If necessary, screeningusing puromycin may be performed to obtain a cell strain with stableexpression of hGPRC6A, and the cell strain may be detected for its geneexpression by qPCR. Experimental results are shown in FIG. 6A. HumanGPRC6A is stably expressed substantially in the selected Hela cells.

S220: experiments of binding of ISAP₂ and hOCN to cell membranes of theHela cells with hGPRC6A overexpressed may be performed.

A method performed herein is relatively the same as the method describedin the above-mentioned embodiment 1. Experimental results are shown inFIG. 6B. Compared with hOCN, the ability of ISAP₂ binding with cellmembranes may be consistent with that of hOCN binding with the cellmembranes, whereas hOCN-22 may not show such ability. Accompanying withthe results of S132 in Embodiment 1, it may be suggested that IASP1 andISAP₂ are the core domains of OCN and hOCN, respectively. IASP1 andISAP₂ may both interact with the receptor hGPRC6A, suggesting that ISAP₁and ISAP₂ may have a same function, leading to subsequent signaling andbiological events through hGPRC6A.

S230: Cy5-labelled OCN, Cy5-labelled hOCN22, and Cy5-labelled ISAP₂ maypromote internalization of GPRC6A in the GPRC6A-overexpressed Helacells.

The hGPRC6A-expressed Hela cell suspension may be plated at a density of1.6×105/ml into a 24-well tray, and each well of the tray may bepre-placed with a gelatin-coated coverslip. 0.5 mL of media containingthe cells may be added into each well, and the cells may be incubatingat 37° C. and 5% CO2 for 24 hours. The cells may be starved for 4 h inserum-free media before any treatment, and then 100 nM of Cy5-OCN, 100nM of Cy5-hOCN22, and 100 nM of Cy5-ISAP₂ may be added to each wellrespectively to incubate with the cells at 37° C. for 30 minutes. Thecells may be fixed by polyoxymethylene for 30 minutes. Afterwards,Triton X may be added to incubate with the cells for 10 minutes. Thecells may be stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma)for 10 seconds as per manufacturer's instructions, and observed andphotographed by a fluorescence confocal microscope. Experimental resultsare shown in FIG. 6C. It can be shown that Cy5-OCN and Cy5-ISAP₂ may bedistributed inside the cell, whereas Cy5-hOCN22 may be distributedextracellularly. It is again suggested that both Cy5-OCN and Cy5-ISAP₂may bind to the receptor and enter the cell through internalization,thereby playing a role in regulating energy metabolism.

S240: effect of gavage of ISAP₂ on HFD mice may be studied.

Six-week-old male wild-type C57BL/6 mice may be divided into 6 groups, 6in each group. Group #1 may be provided with ND to be a ND controlgroup, and groups #2 to #6 may be provided with HFD. Group #2 may betreated as a HFD control group. Groups #3 to #6 may be treated asexperimental groups and may be gavaged with OCN, ISAP₁, ISAP₂, and ISAP₃respectivelyat 2 pmol/g of body weight daily for 7 weeks. Body weightmay be monitored and measured once a week. Experimental results areshown in FIG. 7A. It is apparent from the figure that a significantweight loss occurred for the mice in the ISAP₂-treated group as comparedwith the HFD control group.

Feces of the mice may be collected in the 7th week of the experiment andbaked at 60° C. for 3 days to ensure complete dryness. Then, 1 mg of thefeces may be weighed and soaked in 1 ml of a mixed solution containingchloroform and methanol in a ratio of 2:1. The feces with the mixedsolution may be disrupted by a tissue homogenizer and centrifuged toobtain a supernatant. The supernatant may be detected for triglycerideby the Roche blood biochemical analyzer. Experimental results are shownin FIG. 7B. Compared with the HFD control group, a level of triglyceridein the feces of the ISAP₂-treated mice may be significantly increased,indicating that gavage of ISAP₂ may significantly reduce the absorptionof triglyceride in the intestinal tracts.

Feces of the mice may be collected in the 7th week of the experiment andbaked at 60° C. for 3 days to ensure complete dryness. Then, 1 mg of thefeces may be weighed and soaked in 1 ml of a mixed solution containingchloroform and methanol in a ratio of 2:1. The feces with the mixedsolution may be disrupted by a tissue homogenizer and centrifuged toobtain a supernatant. The supernatant may be detected for triglycerideby the Roche blood biochemical analyzer. Experimental results are shownin FIG. 7B. Compared with the HFD control group, a level of triglyceridein the feces of the ISAP₂-treated mice may be significantly increased,indicating that gavage of ISAP₂ may significantly reduce the absorptionof triglyceride in the intestinal tracts.

Embodiment 3

Study of ISAP₃ Functions

S310: effect of gavage of ISAP₃ on HFD mice may be studied.

Six-week-old male wild-type C57BL/6 mice may be divided into 6 groups, 6in each group. Group #1 may be provided with ND to be a ND controlgroup, groups #2 to #6 may be provided with HFD. Group #2 may be treatedas a HFD control group. Groups #3 to #6 may be treated as experimentalgroups, and may be gavaged with OCN, ISAP₁, ISAP₂, and ISAP₃respectively at 2 pmol/g of body weight daily for 7 weeks. A body weightof each mouse may be monitored and measured once a week. Experimentalresults are shown in FIG. 7A. It is apparent from the figure that asignificant weight loss occurred for the mice in the ISAP₃-treated groupas compared with the HFD control group.

Feces of each mouse may be collected in the 7th week of the experiment,and baked at 60° C. for 3 days to ensure complete dryness. 1 mg of thefeces may be weighed and soaked in 1 ml of a mixed solution containingchloroform and methanol in a ratio of 2:1. The feces in the mixedsolution may be disrupted by a tissue homogenizer and centrifuged toobtain a supernatant. The supernatant may be detected for triglycerideby the Roche blood biochemical analyzer. Experimental results are shownin FIG. 7B. Compared with the HFD control group, a level of triglyceridein the feces of the mice in the ISAP₃-treated group may be significantlyincreased, indicating that gavage of ISAP3 may significantly reduce theabsorption of triglyceride in the intestinal tracts.

Amino acid sequences of ISAP₁, ISAP₂, and ISAP₃ may be compared. ISAP₂may have 4 insertions, 2 substitutions, and 1 deletion of amino acidresidues, compared with the amino acid sequence of ISAP₁. Amino acidsequences of ISAP₃ may have 4 insertions and 3 substitutions of aminoacid residues, as compared with the amino acid sequence of ISAP₁. ISAP₁may have 4 deletions, 2 substitutions, and 1 insertion of amino acidresidues, as compared with ISAP₂. Therefore, the three peptides can beconsidered as variants of each other. It may be presumed that, the threesequences may be used as basis, and amino acid substitutions,insertions, and deletions known to those skilled in the art can beperformed to the three sequences, with the proviso that the ability ofthe peptide to regulate energy metabolism is not significantly reduced,such as by no more than 40%, 30%, 20%, or 10%. Referring to FIG. 8,homologous sequences from a variety of biological sources may becompared. Minimal difference may be found among SEQ ID NO. 2:YLYQWLGAPVPYPDPLEP, SEQ ID NO. 4: YLNNGLGAPAPYPDPLEP, SEQ ID NO. 5:YLYQWLGAPVPYPDTLEP, SEQ ID NO. 6: YLYQWLGAPVPYPDPLEP, SEQ ID NO. 7:YLDHWLGAPAPYPDPLEP, SEQ ID NO. 8: YLDPGLGAPAPYPDPLEP, SEQ ID NO.9:YLDHGLGAPAPYPDPLEP, SEQ ID NO. 10: YLDQGLGAPAPAPDPLEP, and SEQ ID NO.11: YLDSGLGAPVPYPDPLEP. In most cases, when comparing every twosequence, the difference may be no more than four amino acidsubstitutions. It is therefore presumed that peptides having theabove-listed sequences may have similar biological functions and shouldalso be within the scope of the present disclosure. Preferably, thetotal number of deletions, substitutions and insertions may not exceed4, such as no more than 3, 2, or 1. Further, SEQ ID NO. 1 has only oneamino acid residue deleted as compared with SEQ ID NO. 17, so it may beinferred that SEQ ID NO. 1 has functions similar to SEQ ID NO 17.

Further, compared with ISAP₂, ISAP₁ can be seen as having one amino acidinsertion at an end of ISAP₂, and ISAP₃ can also be seen as having oneamino acid insertion at an end of ISAP₂. It may be indicated thatseveral amino acid residues can be added at the end of the functionalpeptide, as long as the ability of modified peptides to regulate energymetabolism is not significantly reduced, for example, by no more than40%, 30%, 20%, or 10%. Preferably no more than 5 amino acid residues,for example 4, 3, 2, 1 or 0 amino acid residues, may be added at the endof the functional peptide.

Embodiment 4

Functions of ISAP₄, ISAP₅, and ISAP₆

S410: effects of acute gavage of ISAP₄, ISAP₅, and ISAP₆ on intestinalabsorption of fat in normal mice may be studied.

15 male wild-type C57BL/6 mice of 6 weeks old may be divided into 5groups, 3 in each group. There may be 3 experimental groups, and themice in the 3 experimental groups may be administered with ISAP₄, ISAP₅,and ISAP₆ at 2 pmol/g of body weight respectively. The mice in twocontrol groups may be gavaged with an equal volume of normal salinedaily for one week. On the 8th day of the experiment, 30 minutes aftergavage of ISAP₄, ISAP₅, ISAP₆, and normal saline respectively, the micein the three experimental groups may be gavaged with 200 μl ofsterilized olive oil, the mice in one of the control groups may also beintragstrically administered with 200 μl of sterilized olive oil to be asaline control group, and the mice in the other one of the controlgroups may further be gavaged with 200 μl of normal saline to be anegative control group. After 50 minutes, each mouse may be euthanizedusing 95% CO2 and dissected to obtain small intestines. The jejunum nearthe duodenum may be frozen and sliced, stained with oil red O, andcounterstained with hematoxylin. An oil red O-positive area may bequantified by the ImageJ software under double-blind conditions and aratio of the oil red O-positive area to a total area of the intestinalvilli may be calculated. Statistical analysis may be performed tocompare with the mince in the saline+olive oil group. ***: p<0.001. N=3.

Experimental results are shown in FIG. 9. The oil red O-positive areamay be quantified by the ImageJ software under double-blind conditionsand the ratio of the oil red O-positive area to the total area of theintestinal villi may be calculated. It can be seen that, compared withthe mice in the saline control group, ISAP₄, ISAP₅, and ISAP₆ mayeffectively reduce the absorption of olive oil in the mice,demonstrating that biological functions of ISAP₄, ISAP₅, and ISAP₆ maybe comparable to those of ISAP₁, ISAP₂, and ISAP₃. Meanwhile, referringto the sequence comparison shown in FIG. 8, it can be seen that SEQ IDNO. 12: PVPYPDPLEP, SEQ ID NO. 15: SVPSPDPLEP, SEQ ID NO. 13: PYPDPLEPand SEQ ID NO. 16: PSPDPLEP are highly similar in sequence, and thesesequences may be very conservative in various species. Therefore, theyshould have similar biological activities, such that these peptides andvariants thereof may be considered to have an effect on the absorptionand metabolism of fat by oral administration. That is to say, amino acidsubstitutions, insertions and deletions well known to those skilled inthe related art can be performed to the sequences, with the proviso thatthe ability of modified peptides to regulate energy metabolism is notsignificantly reduced, for example, by no more than 40%, 30%, 20%, or10%. Moreover, ISAP₄, ISAP₅, and ISAP₆ have shorter sequences, so costsof production may be reduced, and ISAP₄, ISAP₅, and ISAP₆ may exhibitbetter stability, and have great potential for the preparation of drugsfor treating diseases associated with abnormal fat metabolism.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety, as if each individual publication or patentis specifically and individually incorporated by reference. In case ofconflict, the present application, including any definitions herein,shall prevail.

Although some specific embodiments of the present disclosure have beenclearly disclosed herein, the above specification is illustrative ratherthan restrictive. For those skilled in the art, many variations of thepresent disclosure will be apparent by reading the description andappended claims. The full scope of the present disclosure should bedetermined by referring to the claims, the full scope of theirequivalents, and the description and such variations.

What is claimed is:
 1. A peptide for regulating fat metabolism, whereinthe peptide is a variant of a SEQ ID NO: 2 having an amino acid sequenceof YLYQWLGAPVPYPDPLEP, the variant comprises peptides selected from thegroup consisting of: (a) a first amino acid sequence consisting of nomore than 5 amino acids inserted to a C-terminal of the SEQ ID NO: 2,and no more than 5 amino acids inserted to a N-terminal of the SEQ IDNO: 2; (b) a second amino acid sequence consisting of a modified SEQ IDNO: 2, no more than 5 amino acid sequence inserted to a C-terminal ofthe modified SEQ ID NO: 2, and no more than 5 amino acid sequenceinserted to a N-terminal of the modified SEQ ID NO: 2, wherein themodified SEQ ID NO: 2 is obtained by: deleting the Y (Tyr) at a position3 of the SEQ ID NO: 2 or substituting the position 3 of the SEQ ID NO: 2to be Asn or Asp; deleting the Q (Gln) at a position 4 of the SEQ ID NO:2 or substituting the position 4 of the SEQ ID NO: 2 to be Asn, His,Pro, or Ser; deleting the W (Trp) at a position 5 of the SEQ ID NO: 2 orsubstituting the position 5 of the SEQ ID NO: 2 to be Gly; deleting theL (Leu) at a position 6 of the SEQ ID NO: 2; substituting the P (Pro) ata position 9 of the SEQ ID NO: 2 to be Ser; substituting the V (Val) ata position 10 of the SEQ ID NO: 2 to be Ala; substituting the Y (Tyr) ata position 12 of the SEQ ID NO: 2 to be Ser; substituting the P (Pro) ata position 15 of the SEQ ID NO: 2 to be Thr; and a total number ofpositions being substituted and deleted in the second amino acidsequence defined in (b) is less than 4; and (c) a third amino acidsequence consisting of at least 6 contiguous amino acids of the SEQ IDNO: 2 or the modified SEQ ID NO:
 2. 2. The peptide according to claim 1,wherein the peptide comprises an amino acid sequence selected from thegroup consisting of: SEQ ID NO. 1: YLGASVPSPDPLEP; SEQ ID NO .2: YLYQWLGAPVPYPDPLEP; SEQ ID NO. 4: YLNNGLGAPAPYPDPLEP; SEQ ID NO. 5:YLYQWLGAPVPYPDTLEP; SEQ ID NO. 6: YLYQWLGAPVPYPDPLEP; SEQ ID NO. 7:YLDHWLGAPAPYPDPLEP; SEQ ID NO. 8: YLDPGLGAPAPYPDPLEP; SEQ ID NO. 9:YLDHGLGAPAPYPDPLEP; SEQ ID NO. 10: YLDQGLGAPAPAPDPLEP; andSEQ ID NO. 11: YLDSGLGAPVPYPDPLEP.


3. The peptide according to claim 2, wherein a threonine is inserted tothe C-terminal of the SEQ ID NO:1 to obtain a peptide of SEQ ID NO:17(YLGASVPSPDPLEPT).
 4. The peptide according to claim 1, wherein theamino acid sequence of the peptide comprises contiguous residues PDPLEP(SEQ ID NO: 14), and a total number of the amino acids is less than 18.5. The peptide according to claim 4, comprising an amino acid sequenceselected from the group consisting of: EQ ID NO. 12: PVPYPDPLEP;SEQ ID NO. 13: PYPDPLEP; SEQ ID NO. 14: PDPLEP; SEQ ID NO. 15:SVPSPDPLEP; and SEQ ID NO. 16: PSPDPLEP.


6. The peptide according to claim 1, wherein an arginine is inserted tothe N-terminal of the SEQ ID NO: 2 to obtain a peptide of SEQ ID NO: 3(YLYQWLGAPVPYPDPLEPR).